Mechanisms and regulation of replication, the cell cycle, gene expression, and horizontal gene transfer in prokaryotes, focusing on Bacillus subtilis

Massachusetts Institute Of Technology, Cambridge MA

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Abstract

Project Summary/Abstract Horizontal gene transfer (HGT) is a driving force in microbial evolution. It is largely mediated by mobile genetic elements, including viruses, conjugative plasmids, and integrative and conjugative elements (ICEs; aka conjugative transposons), and many bacterial genomes contain several mobile genetic elements, including ICEs and temperate phages. Conjugative elements are well known agents that contribute to the spread of genes for antibiotic resistances, virulence, symbiosis, metabolic functions, and more. ICEs were first discovered because they confer some of these phenotypes. However, potential phenotypes conferred to bacteria by the vast majority of ICEs are not known. It is now clear that ICEs can confer beneficial phenotypes that extend well beyond those of some of the initially characterized ICEs, and that some of these phenotypes involve functional interactions between ICEs and bacterial viruses. It is also apparent that some ICEs, when activated, cause growth arrest of their host bacteria. Despite the prevalence and importance of ICEs, there are major deficiencies in our understanding of these elements, especially in Gram-positive bacteria. Notably, little is known about the interactions between ICEs and their host cells including with co-resident viruses, and the effects ICEs have on fitness of their bacterial hosts, especially at a mechanistic level. Furthermore, little is known about the interactions between functions encoded by ICEs and those encoded by hosts, and how these interactions influence the host range and efficiencies with which ICEs function in different species. ICEs typically reside integrated in the host genome, and the transfer functions are generally not expressed in the vast majority of cells in a population. However, a small subpopulation of cells (typically ~1% or less) contain an active ICE that is expressing genes needed for conjugation. Assays of bulk populations are often not sufficient for detecting effects on the small number of cells with an active ICE. Instead, assays and measurements of individual cells in the active subpopulation are required to determine phenotypes caused by an activated ICE. Visualization of these cells by fluorescence and time-lapse microscopy is a critical tool for these analyses. These types of single-cell analyses are also useful for monitoring gene expression and the change in the cellular location of repair and recombination proteins in response to stresses caused by mobile genetic elements, DNA damage (and other perturbations). Our work will continue to focus on the lifecycle of select ICEs, including ICEBs1 and Tn916, from Gram-positive bacteria. Visualizing events in single cells and small subpopulations will allow us to answer previously difficult or unstudied problems fundamental to the ICE lifecycle and their effects on host cells. Our expertise in chromosome dynamics, DNA replication, stress responses, and microbial development dovetails nicely with our studies of ICEs and phages, notably how these processes affect the lifecycles of mobile genetic elements and how mobile genetic elements affect these processes. Our findings should be relevant to the biology of many bacterial species, especially regarding the transfer of genes between bacteria growing in different environments, including the human microbiome.